Potyvirus屬病毒之廣效性單株抗體、單鏈抗體與其表位以及蕪菁嵌紋病毒感染性選殖株致病因子之研究

Abstract

Potyvirus屬病毒為重要的植物病毒屬之一，且根據ICTV正式命名的病毒數目已多達158種。此屬病毒能夠經由蚜蟲或機械傳播，而其中某些病毒的寄主廣泛，造成嚴重的經濟損害。在台灣，海芋被報導遭受五種potyvirus感染，包括海芋潛徵病毒(Calla lily latent virus, CLLV)、芋頭嵌紋病毒(Dasheen mosaic virus, DsMV)、蒟蒻嵌紋病毒(Konjak mosaic virus, KoMV)、蕪菁嵌紋病毒(Turnip mosaic virus, TuMV)與海芋微嵌紋病毒(Zantedeschia mild mosaic virus, ZaMMV)病毒。為了減少病毒檢定的費用與時間，在先前的實驗中，我們融合並表現DsMV、KoMV與ZaMMV鞘蛋白之121個高保守性胺基酸作為抗原，以其來製備並篩選針對Potyvirus屬病毒的廣效性單株抗體。所篩選出的C4單株抗體除了對於五種海芋病毒皆有不錯的結合力外，還能夠辨識其他九種Potyvirus屬病毒。在本研究的第一部分，我們選殖了C4單株抗體的變異區，並將其建構為C4單鏈抗體(scFv)。在大腸桿菌表現系統中，我們利用新的外泌訊息胜肽(PelE)來促進單鏈抗體的外泌；結果顯示長型或短型的PelE外泌訊息胜肽，皆能夠將C4單鏈抗體外泌到培養基中，並減少無功能內涵體的形成。從西方墨點分析與ELISA結果可知，C4單鏈抗體與C4單株抗體有相似的專一性。第二部分研究，為了尋找C4單株抗體所辨識之表位(epitope)，我們利用噬菌體展現胜肽庫(phage display peptide library)來篩選可被C4單株抗體結合的胺基酸序列。再經由序列分析比對，發現Potyvirus屬病毒之鞘蛋白中的12個保守性胺基酸(WxMMDGxxQxxY/F)可能為C4單株抗體所辨識之表位，故命名為C4表位(C4 epitope)。而點突變的結果顯示色胺酸(W)與酪胺酸(Y)為C4表位與C4單株抗體辨識之重要胺基酸。經由比對C4單株抗體無法辨識之孤挺花嵌紋病毒(Hippeastrum mosaic virus, HiMV)鞘蛋白序列，以及點突變實驗，發現天門冬胺酸(D)亦涉及與C4單株抗體的結合。這些結果顯示C4表位應為Potyvirus屬病毒之鞘蛋白的共同表位。我們亦嘗試將此表位發展成為一個新的表位標籤(epitope tag)。在大腸桿菌表現系統中，我們將齒舌蘭輪斑病毒(Odontoglossum ringspot virus, ORSV)鞘蛋白之C端分別接上ZaMMV、KoMV與DsMV的表位序列，並進行表現與純化。依據西方點墨分析(Western blotting)與酵素連結抗體免疫法(ELISA)的結果，KoMV之序列與C4單株抗體有最高的結合力。我們將此表位序列命名為Ko tag，並用於植物與哺乳類動物細胞系統。在植物中短暫表現接上Ko tag的GFP與ORSV 鞘蛋白，兩者皆可以被C4單株抗體所辨識；而Ko tag不會影響蕃茄叢生矮化病毒(Tomato bushy stunt virus, TBSV)之P19 (基因靜默抑制子)之基因靜默能力。在哺乳類動物細胞系統中，短暫表現接上Ko tag的eGFP亦可以被C4單株抗體所辨識，並且可利用C4單株抗體進行EGFP的免疫沉澱。這些結果證明在細菌、植物和哺乳類動物細胞系統中，Ko tag具有成為一個新表位標籤的潛力。第三部分研究，為了建立potyvirus病毒之反向遺傳系統，我們以蕪菁嵌紋病毒(TuMV)作為材料，以兩步驟選殖的方式得到了四個TuMV的全長選殖株。在不同寄主的接種實驗中，p35S-TuMV-1與p35S-TuMV-27選殖株皆比p35S-TuMV-5與p35S-TuMV-6選殖株有較好的感染力。而經過全長定序後，我們發現TuMV-5、TuMV-6與TuMV-27只在五端非轉譯區有一至三個核苷酸的差異。而在五端非轉譯區的置換與點突變實驗中，我們確認缺少第七個腺嘌呤核苷酸會造成其接種成功率下降。另外，我們利用TuMV-27感染性選殖株來分析為何另一個全長選殖株pTuMV-T100會缺乏感染力。我們將pTuMV-T100的基因體依照不同的限制酶切位分為三個片段(AB, BH與HX)，並置換到TuMV-27之中。接種實驗結果顯示這三個片段皆含有會影響感染力的點突變，其中TuMV-AB與TuMB-BH只能在單細胞層次複製，但卻無法成功感染菸草與白藜，顯示其點突變可能影響到其在細胞間移動的能力 。而帶有兩個鞘蛋白點突變的TuMV-HX接種白藜只能夠產生較小病斑；在菸草單細胞中，其鞘蛋白的累積量則遠低於TuMV-27。利用免疫沈澱和專一性單株抗體，我們證明TuMV鞘蛋白的酪胺酸被磷酸化。進一步利用點突變分析，顯示TuMV鞘蛋白的第219位置的酪胺酸突變(Y219N)，會造成鞘蛋白累積量下降如同TuMV-HX。同樣的，另外兩個酪胺酸突變株(TuMV-Y191A與TuMV-Y224A)的接種結果與TuMV-Y219N類似。而在植物中短暫表現TuMV鞘蛋白的實驗顯示，酪胺酸突變的鞘蛋白其穩定性較野生型的鞘蛋白為差。根據這些結果，我們推測TuMV鞘蛋白的穩定性可能與酪胺酸的磷酸化相關。

The genus Potyvirus is one of the important plant virus genera, and comprises 158 formal species based on ICTV Virus Taxonomy. Potyviruses can be transmitted by aphids and mechanical inoculation. Some potyviruses have broad host range and cause serious economic losses. In Taiwan, calla lily is reported to be infected by five potyviruses, Calla lily latent virus (CLLV), Dasheen mosaic virus (DsMV), Konjak mosaic virus (KoMV), Turnip mosaic virus (TuMV), and Zantedeschia mild mosaic virus (ZaMMV). To reduce the cost and time of virus indexing, the conserved 121-amino-acids core regions of the capsid protein (CP) of DsMV, KoMV, and ZaMMV were concatenated and expressed. The recombinant protein was used as an antigen to prepare and screen the potyvirus group-specific monoclonal antibody (MAb). The selected C4 MAb could detect nine potyviruses in addition to the five calla lily potyviruses. In the first part of this study, we cloned the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb, and then constructed them as C4 single-chain variable fragments (scFvs). In E. coil expression system, a new PelE secretory signal peptide was used to help the secretion of C4 scFv. The data showed not only long but also short PelE signal peptide could secrete C4 scFv to medium and reduce inclusion body formation. According to western blot and I-ELISA, the soluble C4 scFv showed a binding specificity similar to that of the C4 MAb. In the second part, to identify which epitope is recognized by C4 MAb, the phage display peptide library was used to screen the C4 MAb-reactive peptides. The sequence alignment of C4 MAb-reactive peptides with potyviral CP sequences indicated that a conserved 12-amino acid (WxMMDGxxQxxY/F) sequence may be recognized by C4 MAb, and thus it was named as the C4 epitope. The results of amino acid substitution analysis indicated that tryptophan and tyrosine residues of C4 epitope are crucial for reacting with C4 MAb. Furthermore, sequence alignment of Hippeastrum mosaic virus (HiMV), which could not be detected by C4 MAb, and amino acid substitution analysis also showed the aspartic acid is also involved in binding with C4 MAb. These results of epitope mapping demonstrated the C4 epitope is a common CP epitope of potyviruses. We also tried to develop the C4 epitope as a new epitope tag. The epitope sequences of ZaMMV, KoMV, and DsMV were separately fused to the C-terminus of CP of Odontoglossum ringspot virus (ORSV), and then epitope-tagged ORSV CPs were expressed in a bacterial system and purified. The results of Western blotting and ELISA showed the C4 epitope of KoMV (Ko) had the strongest binding affinity to C4 MAb. To examine the applicability of Ko tag in planta, the transiently expressed Ko-tagged GFP and ORSV CP could be successfully detected by C4 MAb, and the Ko-tagged P19 of Tomato bushy stunt virus (TBSV) still maintained its silencing suppressor function. Furthermore, Ko-tagged EGFP could be successfully detected and subsequently immunoprecipitated by C4 MAb in a mammalian cell system. These data proved that Ko tag has the potential to become a new epitope tag in bacterial, plant, mammalian cell systems. In the third part, TuMV was used as an experimental material to develop a reverse genetic system. We obtained four full-length clone of TuMV by a two-step cloning method. In the infectivity assay, the p35S-TuMV-27 clone which had similar infectivity to p35S-TuMV-1 revealed much better infectivity than p35S-TuMV-5 and p35S-TuMV-6. The sequence comparison of TuMV-5, TuMV-6 and TuMV-27 clones indicated that they have only 1~3 nucleotide difference at the extreme 5' end of viral genome. After 5’ replacement and 5’ G deletion analyses, these data verified that the lacking of the adenine at position 7 of 5’ UTR could reduce the infectivity. Besides, the infectious TuMV-27 clone was used to analyze which amino acid residues responsible for the lack of infectivity of pTuMV-T100 clone. Based on unique restriction enzyme sites, three fragments (AB, BH, and HX) of TuMV-T100 were used to separately replace the corresponding fragments of TuMV-27. The result of infectivity assay indicated all of these fragments contain the amino acid mutations affected infectivity. TuMV-AB and TuMV-BH could replicate in protoplasts, but could not infect N. benthamiana and C. quinoa plants. These results indicated that they may be defective in cell-to-cell movement. In contrast, TuMV-HX could induce small local lesions on C. quinoa, but its CP accumulation was lower than that of TuMV-27. The point mutation assay confirmed that CP mutation of Y219N decreased the infectivity of TuMV-27. In addition, other tyrosine mutants (TuMV-Y191A, TuMV-Y219A, and TuMV-Y224A) had similar phenotype as TuMV-Y219N. In transient expression assay, the tyrosine-mutated CP was less stable than wild-type CP. Thus, we suggested that the stability of TuMV CP may be affected by tyrosine phosphorylation.